Various publications, which may include patents, published applications, technical articles and scholarly articles, are cited throughout the specification in parentheses, and full citations of each may be found at the end of the specification. Each of these cited publications is incorporated by reference herein, in its entirety.
Matrix metalloproteinases (MMPs) are a family of structurally related zinc-dependent proteolytic enzymes that digest extracellular matrix proteins such as collagen, elastin, laminin and fibronectin. Currently, at least 28 different mammalian MMP proteins have been identified and they are grouped based on substrate specificity and domain structure. Enzymatic activities of the MMPs are precisely controlled, not only by their gene expression in various cell types, but also by activation of their inactive zymogen precursors (proMMPs) and inhibition by endogenous inhibitors and tissue inhibitors of metalloproteinases (TIMPs). The enzymes play a key role in normal homeostatic tissue remodeling events, but are also considered to play a key role in pathological destruction of the matrix in many connective tissue diseases such as arthritis, periodontitis, and tissue ulceration and also in cancer cell invasion and metastasis.
A role for MMPs in oncology is well established, as up-regulation of any number of MMPs are one mechanism by which malignant cells can overcome connective tissue barriers and metastasize (Vihinen, Ala-aho et al. 2005). MMPs also appear to have a direct role in angiogenesis, which is another reason they have been an important target for oncology indications (Handsley and Edwards 2005; Rundhaug 2005). Several different classes of MMPs are involved in these processes, including for example MMP9, MMP2, and MT1-MMP.
Other MMP mediated indications include the cartilage and bone degeneration that results in osteoarthritis and rheumatoid arthritis. The degeneration is due primarily to MMP digestion of the extracellular matrix (ECM) in bone and joints (Iannone and Lapadula 2003). MMP1, MMP3, MMP9, and MMP13 have all been found to be elevated in the tissues and body fluids surrounding the damaged areas.
MMPs may also have a role in cardiovascular diseases, in that they are believed to be involved in atherosclerotic plaque rupture, aneurysm and vascular and myocardial tissue morphogenesis (George 2000; Tayebjee, Lip et al. 2005). Elevated levels of MMP1, MMP2, MMP9, and MMP13 have often been associated with these conditions. Several other pathologies such as gastric ulcers, pulmonary hypertension, chronic obstructive pulmonary disease, inflammatory bowel disease, periodontal disease, skin ulcers, liver fibrosis, emphysema, and Marfan syndrome all appear to have an MMP component as well (Shah, Wilkin et al. 2002).
Within the central nervous system, altered MMP expression has been linked to several neurodegenerative disease states (Yong 1999), most notably in stroke (Cunningham, Wetzel et al. 2005). In particular, MMP2 and MMP9 appear to have the significant impact in propagating the brain tissue damage that occurs following an ischemic or hemorrhagic insult. Studies in human stroke patients and in animal stroke models have demonstrated that both MMP2 and MMP9 expression levels and activity increase sharply over a 24 hour period following an ischemic event. Within the brain, the microvascular endothelial cell tight junctions are broken down by activated MMP2 and MMP9, which results in increased permeability of the blood-brain barrier (BBB). This breakdown in the integrity of the BBB then leads to edema and infiltration of inflammatory agents, both of which cause increased cell death around the infarct core (the penumbra) and increase the possibility of hemorrhagic transformation. Administration of MMP inhibitors has been shown to be protective in animal models of stroke (Yong 1999; Gu, Cui et al. 2005). In addition, MMP9 knockout animals also demonstrate significant neuroprotection in similar stroke models (Asahi, Asahi et al. 2000). In the US, stroke is the third leading cause of mortality, and the leading cause of disability. Thus this area has a large unmet medical need for acute interventional therapy that could potentially be addressed with MMP inhibitors.
It has also been suggested that MMP9 may play a role in the progression of multiple sclerosis (MS). Studies have indicated that serum levels of MMP9 are elevated in active patients, and are concentrated around MS lesions (Opdenakker, Nelissen et al. 2003). Increased serum MMP9 activity would promote infiltration of leukocytes into the CNS, a causal factor and one of the hallmarks of the disease. MMPs may also contribute to severity and prolongation of migraines. In animal models of migraine (cortical spreading depression), MMP9 is rapidly upregulated and activated leading to a breakdown in the BBB, which results in mild to moderate edema (Gursoy-Ozdemir, Qiu et al. 2004). It is this brain swelling and subsequent vasoconstriction which causes the debilitating headaches and other symptoms associated with migraine. In the cortical spreading depression model, MMP inhibitors have been shown to prevent the opening of the BBB (Gursoy-Ozdemir, Qiu et al. 2004). Related research has shown that MMP9 is specifically upregulated in damaged brain tissues following traumatic brain injury (Wang, Mori et al. 2002), which would be predicted to lead to further brain damage due to edema and immune cell infiltration. MMPs may also have additional roles in additional chronic CNS disorders. In an animal model of Parkinson's disease, MMP9 was found to be rapidly upregulated after striatal injection of a dopaminergic neuron poison (MPTP) (Lorenzl, Calingasan et al. 2004), and MMP3 has been shown to process α-synuclein to an aggregation-prone form (Sung, Park et al. 2005). This implicates MMPs in both the neuronal remodeling that occurs upon cell loss and one of the potential causative factors of the disease. In patients with Alzheimer's disease, MMP9 was found to be upregulated in postmortem plasma samples compared to normal controls (Yong 1999; Lorenzl, Albers et al. 2003). Furthermore, pathologic expression of amyloid beta peptides induces expression and activation of MMP2, which may contribute to cerebral amyloid angiopathy, a major pathological feature of Alzheimer's disease (Jung, Zhang et al. 2003). MMPs may also have a role in vascular dementia, as MMP9 levels have been found to be elevated in the cerebrospinal fluid from demented patients (Adair, Charlie et al. 2004).
With regard to structure and activation of the inactive zymogen form, a prototypical MMP is matrix metalloproteinase 9 (MMP9). MMP9 is also known as macrophage gelatinase, gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase, and type V collagenase. The inactive form of MMP9, proMMP9, is expressed with several different domains including a signal sequence for secretion, a propeptide domain which inhibits activity of proMMP9, a catalytic domain for protein cleavage, a fibronectin type-II (FnII) domain consisting of three fibronectin-type II repeats, and a hemopexin-like domain thought to assist in substrate docking. The hemopexin-like domain also serves as a binding domain for interaction with Tissue Inhibitors of Metaloproteinases (TIMPs). The inactive zymogen form of MMP9, proMMP9, is maintained through a cysteine-switch mechanism, in which a Cys in the propeptide forms a complex with the catalytic zinc in the catalytic domain and occludes the active site (Van Wart and Birkedal-Hansen 1990). Activation of proMMP9 occurs in a two-step process. A protease cleaves an initial site after Met 60, disrupting the zinc coordination and destabilizing the propeptide interaction with the catalytic domain. This initial cleavage allows access to the second cleavage site at Phe 107, after which the propeptide is removed and the mature active form of the enzyme is released (Nagase 1997). The identity of the MMP9 activating proteases is unknown in vivo, although there is evidence that activation can occur through the actions of MMP3, chymase and trypsin (Ogata, Enghild et al. 1992; Fang, Raymond et al. 1997; Tchougounova, Lundequist et al. 2005).
Crystal structures of MMP9 and proMMP9 have been reported. A structure of the C-terminally truncated proMMP9 was reported to 2.5 Å resolution (Elkins, Ho et al. 2002). The structure contained the pro domain, the catalytic domain and the fibronectin-type II (FnII) repeats, but the structure did not contain active site inhibitors or allosteric processing inhibitors. Two additional publications reported the structure of the catalytic domain of MMP9 without the FnII repeats (Rowsell, Hawtin et al. 2002; Tochowicz, Maskos et al. 2007). The structures of the MMP9 catalytic domain showed both the apo and active site inhibited forms of the protein. The structures solved to date show a high degree of structural homology. No large difference in structure was noted due to the presence or lack of the FnII repeats. No structure reported to date identifies compounds binding to the region near residue Phe 107. In addition to the proMMP9 structure, the structures of proMMP1 (Jozic, Bourenkov et al. 2005), proMMP2 (Morgunova, Tuuttila et al. 1999), and proMMP3 (Becker, Marcy et al. 1995) have also been reported.
Based on the demonstrated involvement in numerous pathological conditions, inhibitors of matrix metalloproteases (MMPs) have been widely sought for their therapeutic potential in a range of disease states. However, non-selective active site MMP inhibitors have performed poorly in clinical trials. The failures have often been caused by dose-limiting toxicity and the manifestation of significant side effects, including the development of musculoskeletal syndrome (MSS). It has been suggested that development of more selective MMP inhibitors might help to overcome some of the problems that hindered clinical success in the past, but there are a number of obstacles to developing more selective MMP active site inhibitors. MMPs share a catalytically important Zn2+ ion in the active site and a highly conserved zinc-binding motif. In addition, there is considerable sequence conservation across the entire catalytic domain for members of the MMP family.
Herein is described a novel approach to developing more selective MMP inhibitors by targeting the pro domain of the inactive zymogens, proMMPs, with small-molecule allosteric processing inhibitors that bind and stabilize the inactive pro form of the protein and inhibit processing to the active enzyme. There is significantly less sequence identity within the pro domains of MMP proteins, no catalytically important Zn2+ ion, and no highly conserved zinc-binding motif. Thus targeting the pro domain of proMMPs is an attractive mechanism of action for inhibiting the activity of the MMP proteins Inhibition of proMMP9 activation has been observed with a specific monoclonal antibody (Ramos-DeSimone, Moll et al. 1993). The activation of proMMP9 by trypsin has also been shown to be inhibited by Bowman-Birk inhibitor proteins and derived peptide inhibitors (Losso, Munene et al. 2004). There are no reports, however, of small-molecule allosteric processing inhibitors that inhibit the proteolytic activation of proMMP9 or any other proMMP. The present invention provides methods of identifying such small-molecule allosteric processing inhibitors and methods of treatment using such inhibitors.